The two-spotted spider mite Tetranychus urticae is a worldwide crop pest with a high insecticide resistance and an extensive host range. The aim of the present study was to evaluate the effect of PaeciPora®, which was formulated from the aerial conidia of an entomopathogenic fungus Paecilomyces lilacinus strain HY-4, to control T. urticae in cucumber field. In the field study, conidia of P. lilacinus HY-4 and a chemical acaricide azocyclotin were investigated for their control of the adult females of T. urticae. The strain produced a mortality of 56.0% on day 3 and 63.6% on day 7 post-treatment respectively at 1×107 conidia/mL, and no evidence of a mortality benefit was seen in the control group. Additionally, in the pesticide injury test, no agrochemical damage was found in hot pepper, watermelon, Chinese cabbage, oriental melon or strawberry by spraying PaeciPora® on them. The results indicated the possibility of the use of P. lilacinus HY-4 as a microbiological control agent against T. urticae in the Integrated Pest Management program.

An entomopathogenic filamentous fungus, Paecilomyces lilacinus strain HY-4, has a great potential as a promising bio-pesticide due to its superior pathogenicity against Adoretus tenuimaculatus and Tetranychus urticae. When the fungal strain infects host cuticle, it secrets a combination of hydrolytic enzymes including chitinase to solubilize the cuticle. Thus, we investigated effects of different carbon and nitrogen sources on the production of a chitinase from P. lilacinus strain HY-4. The organism produced an extracellular chitinase at a relatively high level (45.4 mU/ml) when cultivated for 5 days on a medium supplemented with insect pupa (0.5%) and colloidal chitin (1%), which was prepared by treating chitin from crab shells (Sigma-Aldrich Co. Ltd.) with 12 N HCl solution. However, extracellular secretion of chitinase by strain HY-4 was found to be significantly repressed in the presence of glucose (1%).

The extracellular GH11 β-1,4-xylanase (XylY) gene (633-bp) of Paenibacillus sp. strain KYJ-16 was molecularly cloned by repeated DNA walking and nested PCR method. The xylY gene was predicted to encode an extracellular protein consisting of 611 amino acids with a nesuced molecular mass of 23 kDa and a calculated pI of 9.55. Protein blast search revealed that the enzyme consisted of a putative catalytic domain, which is homologous to a catalytic GH11 domain. The highest sequence identity (92%) was obtained as the catalytic GH11 domain of XylY was compared to that of Paenibacillus sp. strain HGF5 (GenBank accession number: EGG35584) that has not yet been characterized. Enzymatic properties of the recombinant His-tagged enzyme (rXylY) overexpressed in E. coli BL21 harboring pET-28a(+)/xylY will be also presented.

The gene (2,304-bp) encoding a novel xylanolytic enzyme (XylD) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 β-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylDΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg-1) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylDΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-β-1,4-xylanase activity of XylDΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50 mM xylobiose.

xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum (LBG), guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManS, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50℃ and pH 7.0, ManK exhibited extraordinary high specific activities of 7,109 IU/mg and 5,158 IU/mg toward LBG and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. ManK strongly attached to Avicel, lignin, β-cyclodextrin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManS suggest that it is a unique endo-β -1,4-mannanase with out additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Paecilomyces lilacinus HY-4 is an entomopathogenic filamentous fungus that has exhibited insecticidal activity against Adoretus tenuimaculatus and Tetranychus urticae. Strain HY-4 has attracted a great deal of industrial concerns because this organism can be applicable as a potent bio-pesticide. In this study, we developed an optimal diphasic fermentation technique for HY-4 conidial production. The substrate prescription which was made up of hulls or brans was obtained by screening of agricultural products, and the conidial production could reach a minimum of 2.5×1010 conidia/g after solid fermentation for 12 days in a given condition. It was interesting to note that strain HY-4 was propitious to sporulate more efficiently and productively in liquid fermentation process in the presence of insect pupa. For the industrial application of the HY-4 spores, their pH and thermal stabilities were evaluated on SDA agar plate as well. Additionally, the germination rate of HY-4 spores was still above 90% even when they were stored for 5 months at ambient temperature.

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen‐thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2‐cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time‐dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.